Mechanisms for the maintenance and eventual degradation of neurofilament proteins in the distal segments of severed goldfish mauthner axons

Abstract

Cellular mechanisms that might affect the degradation of neurofilament proteins (NFPs) were examined in the distal segments of severed goldfish Mauthner axons (M-axons), which do not degenerate for more than 2 months after severance. Calpain levels, as determined by reactivity to a polyclonal antibody, remained constant for 80 d postseverance in distal segments of M-axons and then declined from 80 to 85 d postseverance. Calpain activity in rat brain, as determined by a spectrophotometric assay, was much higher than calpain activity in control and severed goldfish brain, spinal cord, muscle, or M-axons. Calpain activity was extremely low in M-axons compared with that in all other tissues and remained low for up to 80 d postseverance in distal segments of M-axons. Phosphorylated NFPs, as determined by Stains-All treatment of SDS gels, were maintained for up to 72 d postseverance and then decreased noticeably at 75 d postseverance when NFP breakdown products appeared on silver-stained gels. By 85 d post-severance, phosphorylated NFPs no longer were detected, and NFP breakdown products were the most prominent bands on silver-stained gels. These results suggest that the distal segments of M-axons survive for months after severance, because NFPs are maintained in a phosphorylated state that stabilizes and protects NFPs from degradation by low levels of calpain activity in the M-axon; the distal segments of severed M-axons degenerate eventually when NFPs no longer are maintained in a phosphorylated state and become susceptible to degradation, possibly by low levels of calpain activity in the M-axon.

Calpain activity promotes the sealing of severed giant axons

“…(ii) Inhibitors of protease activity some of which are specific for calpain (e.g., calpain antibody, human calpastatin peptide, endogenous rabbit calpastatin) inhibit seal formation in crayfish MGAs. (iii) Calpain is an endogenous protease in squid GAs (5), other axons (including the crayfish MGA) (4,6,8,10,20), and many other cell types (14)(15)(16)(17)(18)(19). (While other proteases may induce sealing in Ca 2ϩ -free salines in crayfish MGAs (this report) and in mammalian axons (11), these proteases are not known to be endogenous to these axons.)…”

“…Calpain and other proteases degrade cytoskeletal proteins such as neurofilaments (4,6,7,8), which help maintain axonal diameter and shape (9). Such degradation might produce a complete or partial collapse of the cut end (10,11), thereby inducing sealing by enhancing the interactions between vesicles that form a seal by themselves and͞or that form a continuous membrane at the cut end. (In this paper, the term ''complete constriction or complete collapse'' is used to describe 100% closure of a cut axonal end; the term ''partial constriction or partial collapse'' is used to describe any constriction that leaves an opening at the cut end.)…”

Neuronal and non-neuronal responses to nerve crush in a pulmonate snail, Melampus bidentatus

“…In the case of the distal stumps of S-cells of the leech Hirudo, survival is documented for 2±5 months unless reinnervation by the proximal stump occurs, whereupon the distal stump quickly disintegrates (Muller and Carbonetto 1979). The process of degeneration of distal segments has been correlated with the degradation of neuro®lament proteins, which occurs rapidly in mammals and very slowly in the gold®sh Mauthner cell axon (Raabe et al 1996). These dierences may re¯ect relative levels of calpain activity, although stimulation of phagocytosis or endogenous lysosomal activity may account for degeneration in other cases.…”

Degeneration, Trophic Interactions, and Repair of Severed Axons: A Reconsideration of Some Common Assumptions

“…Axotomy does not necessarily remove all major sources of trophic support from the distal segment, which can have a major trophic dependence on proteins received from various combinations of adjacent glia, neurons, or axonal protein synthesis (14-16, 25, 37-40). (Slow turnover of existing axonal proteins may also be important [41][42][43][44][45][46].) 3.…”